This small grant application has been prepared by a new investigator in order to obtain the preliminary data necessary to prepare a competitive RO1 grant application on endothelial cell dysfunction in insulin resistant-states. The objective of this application is to identify the regulatory mechanisms involved in insulin-stimulated Na+/Mg2+ exchange activity in cells from normal and diabetic patients. Our long-term goal is to define the cellular mechanisms that lead to insulin resistance and diabetes. The central hypothesis for the proposed research is that low levels of Mg2+ attenuate the intracellular signal generated following insulin binding to its receptor or antagonize binding of insulin to the receptor. This hypothesis is based on preliminary findings obtained in human microvascular endothelial cells and red blood cells. We propose to test our central hypothesis and accomplish the overall objectives of this proposal through the following specific aims: Aim 1: Identify the role of insulin in cellular Mg2+ regulation in human microvascular endothelial cells. On the basis of our preliminary data, we hypothesize that insulin regulates cellular Mg2+ levels via activation of the exchanger, which in turn regulates nitric oxide production in human endothelial cells. We will characterize the intracellular signaling pathways that are downstream of PI3-kinase activation and modulate the activity of the exchanger as well as nitric oxide production in these cells Aim 2: Identify the mechanisms for insulin-regulated Na+/Mg2+ exchange activity in ex vivo human red cells from normal subjects. Erythrocytes have been used as ex vivo models of what may occur in target tissue of insulin resistance's pathophysiology. We hypothesize that the insulin receptor is functionally coupled to the exchanger in human red cells via PI3-kinase activation. Therefore, our studies are designed to characterize PI3-kinase activity as well as phosphorylated Akt levels in erythrocytes upon activation with insulin. Aim 3: Identify the mechanisms for regulation of Na+/Mg2+ exchange activity in ex vivo human red cells from Type 2 diabetes mellitus patients. We hypothesize that elevated Na+/Mg2+ exchange activity in the red cells of diabetic patients in comparison to normal subjects explains the low cellular Mg2+ levels observed in patients with diabetes. This suggests an uncoupling between the insulin receptor and the exchanger in these patients. Therefore, we hypothesize that PI3-kinase activity is altered in diabetic red cells when compared to normal. We will compare PI3-kinase activity and phosphorylated Akt levels in erythrocytes from normal and diabetic subjects. We expect that these studies will identify novel cellular mechanisms underlying insulin resistance as well as characterize cellular Mg2+ regulatory mechanisms.